Cell plasma membranes form the outer surfaces of eukaryotic cells. Various ions (e.g., sodium, potassium, calcium, etc.) move in and out of cells by passive diffusion through the cells' plasma membranes. Such diffusion of ions into and out of cells is facilitated by the presence of “ion channels” within the cell membranes. Ion channels are proteins embedded within the cell membrane that control the selective flux of ions across the membrane, thereby allowing for the formation of concentration gradients between the intracellular contents of the cell and the surrounding extracellular fluid. Because ion concentrations are directly involved in the electrical activity of excitable cells (e.g., neurons), the functioning (or malfunctioning) of ion channels can substantially control the electrical properties and behavior of such cells. Indeed, a variety of disorders, broadly termed “channelopathies,” are believed to be linked to ion channel insufficiencies or dysfunctions.
Ion channels are referred to as “gated” if they can be opened or closed. The basic types of gated ion channels include a) ligand gated channels, b) mechanically gated channels and c) voltage gated channels. In particular, voltage gated channels are found in neurons, muscle cells and non-excitable cells such as lymphocytes. They open or close in response to changes in the charge across the plasma membrane.
Kv1.3 Channels and Autoimmune Diseases.
Autoimmune diseases such as multiple sclerosis (MS), type-1 diabetes mellitus (T1DM), rheumatoid arthritis (RA) and psoriasis affect several hundred million people worldwide. In these disorders specific autoreactive T cells—for instance myelin-specific T cells in MS patients—are believed to undergo repeated autoantigen stimulation during the course of disease and differentiate into chronically activated memory cells that contribute to pathogenesis by migrating to inflamed tissues and secreting cytokines (Viglietta et al., 2002; Vissers et al., 1002; Wulff et al., 2003b). Therapies that preferentially target chronically activated memory T cells would have significant value for autoimmune diseases.
Memory T cells are divided into two subsets—central memory (TCM) and effector memory (TEM)—based on the expression of the chemokine receptor CCR7 and the phosphatase CD45RA (Geginat et al., 2001; Sallusto et al., 1999). Naïve and TCM cells home to the lymph node before they migrate to sites of inflammation, whereas TEM cells home directly to sites of inflammation where they secrete copious amounts of IFN-β and TNF-α and exhibit immediate effector function. It has recently been shown that myelin-specific autoreactive T cells in MS patients are predominantly activated TEM cells (Wulff et al., 2003b), and adoptive transfer of myelin-specific activated rat TEM cells into naïve recipients induced severe EAE (Beeton et al., 2001a; Beeton et al., 2001b). An exciting new therapeutic target for immunomodulation of TEM cells is the voltage-gated Kv1.3 K+ channel. TEM cells up-regulate Kv1.3 channels upon activation and their antigen-driven proliferation is exquisitely sensitive to Kv1.3 blockers (Wulff et al., 2003b). Naïve and TCM cells in contrast are significantly less sensitive to Kv1.3 blockers to begin with and rapidly become resistant to Kv1.3 blockade by up-regulating the calcium-activated K+ channel IKCa1 (Ghanshani et al., 2000; Wulff et al., 2003b).
The dominance of Kv1.3 in TEM cells provides a powerful way to manipulate the activity of this subset with specific Kv1.3 inhibitors. The functionally restricted tissue distribution of the channel and the fact that in vivo Kv1.3 blockade ameliorates TEM-mediated EAE, bone resorption in peridontal disease and delayed type hypersensitivity reactions in animal models without causing obvious side effects has enhanced the attractiveness of Kv1.3 as a therapeutic target (Beeton et al., 2001b, Koo et al., 1997; Valverde et al., 2004). Although Kv1.3 blockers would suppress all activated TEM cells (for example TEM cells specific for vaccine antigens), a Kv1.3-based therapy would be a significant improvement over current therapies that broadly and indiscriminately modulate the entire immune system. An additional advantage of Kv1.3 blockers is that they are reversible. Thus, one could titrate the therapeutic effect of Kv1.3 blockers when needed and stop therapy in the face of infection, unlike chemotherapeutic agents, which take months to subside.
Kv1.3 Channels and Obesity
The Kv1.3 channel was found to play a role in energy homeostasis and energy balance (Hum Mol Genet. 2003 12:551-9). Mice with the Kv1.3 channel genetically knocked out were able to eat fatty diets without gaining weight, while control mice given the same diet became over-weight. Pharmacological blockade of Kv1.3 channels recapitulated the effect of genetic knockout of Kv1.3 channels. Consequently, Kv1.3 blockers are likely to have use in the management of obesity.
Kv1.3 Channels and Type-2 Diabetes Mellitus.
Kv1.3 channels play a role in regulating insulin-sensitivity in peripheral target organs such as the liver and muscle (Proc Natl Acad Sci USA. 2004 101:3112-7). Genetic knockout of the Kv1.3 channel in mice enhanced the sensitivity of the liver and muscle to insulin. Consequently, Kv1.3 blockers may have use in the treatment of type-2 diabetes mellitus by enhancing insulin's peripheral actions and thereby decreasing blood glucose levels.
Naturally Occurring Polypeptides Known to Inhibit Kv1.3 Channels
The most potent Kv1.3 inhibitor is the peptide ShK from the Caribbean sea anemone Stichodactyla helianthus. ShK is a 35-residue polypeptide cross-linked by 3 disulfide bridges. ShK blocks Kv1.3 (Kd=11 pM) and suppresses proliferation of TEM cells at picomolar concentrations, and ameliorates experimental autoimmune encephalomyelitis (EAE) in rats induced by the adoptive transfer of myelin-specific TEM cells. A potential drawback of ShK is its low picomolar affinity for the neuronal Kv1.1 channel (Kd 28 pM). Although no side effects were observed with ShK in EAE trials, ingress of high concentrations of ShK into the brain, as might happen when the blood-brain-barrier is compromised in MS, could lead to unwanted neurotoxicity. The development of highly specific Kv1.3 inhibitors is therefore necessary. An extensive effort by the pharmaceutical industry and academic groups has yielded several small molecules that inhibit Kv1.3 in the mid-nanomolar range, but these compounds do not have the selectivity or potency to make them viable drug candidates.
Several truncated peptidic analogs of ShK have previously been reported. In one of these ShK analogs, the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges). In others, non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. These ShK analogs exhibited varying degrees of Kv1.3 inhibitory activity and specificity. Lanigan, M. D. et al.; Designed Peptide Analogues of the Potassium Channel Blocker ShK Toxin; Biochemistry, 25; 40(51):15528-37 (December 2001).
There remains a need in the art for the development of new analogs of ShK that selectively inhibit Kv1.3 channels in lymphocytes with minimal or no inhibitory effects on Kv1.1 channels or other potassium channels.